PB2480: LENTIVIRAL LABELING OF STROMAL LAYER OF LONG-TERM BONE MARROW CULTURE DEMONSTRATED INEFFICIENCY FOR MESENCHYMAL STEM CELLS AND CONTRIBUTION OF NON-STEM CELLS TO HEMATOPOIETIC ECTOPIC FOCUS IN MICE
Authors: Dmitriy Karpenko, Aleksei Bigildeev
Journal: HemaSphere (2023)

Topic: 23. Hematopoiesis, stem cells and microenvironment Background: Transplantation of a bone marrow (BM) or an adherent cell layer (ACL) of a long-term bone marrow culture (LTBMC) under the kidney capsule of syngeneic recipient lead to hematopoietic ectopic focus formation (HEF). It is a model for studying formation and functioning of BM hematopoietic territory in conditions close to native. It is considered that HEF forms de novo from mesenchymal stem cells (MSCs), while differentiated progeny does not survive the procedure. It was also mentioned earlier that transduction of LTBMC by lentiviral vectors effectively marks MSCs. Aims: We used these methods to assess the peculiarities of lentiviral transduction of BM mesenchymal cells and to study their clonal activity in the process of HEF formation. Methods: ACL of each LTBMC (n = 10) from CBF1 mouse BM were transduced with a self-inactivating LeGO lentiviral vector based on HIV-1. Random site of integration (SI) of a provirus provided genetically inherited marker unique for each initial integration. Transduced ACLs were implanted under the kidney capsule in syngeneic recipients for 42 days. To evaluate labeling efficiency of stromal cells, 15000 cells/well (n = 540) were seeded in 96-well plates from each HEF’s inner cell mass (ICM). Two weeks later, DNA was isolated from each well and provirus-containing wells were determined by traditional nested PCR (tnPCR) and digital droplet PCR (ddPCR). Libraries for SIs sequencing on Illumina MiSeq (NGS) were prepared from DNA of HEFs using PCR. Bioinformatics analysis was performed using the original algorithm in the Python, SI mapping was performed using the NCBI BLAST. Results: Despite high efficiency of ACLs transduction, only 33/309 wells were labeled out of 309 wells with marked cell growth according to tnPCR. Only 8/33 of those samples contained 0.4-7.1% marked cells (1-3 marker molecules per sample) according to ddPCR: totally, there were 12 provirus DNA molecules registered for 10389 of genomic Gapdh molecules. NGS analysis showed that only 15 out of 2165 analyzed clones among all samples consisted of ≥ 6 cells. Therefore, cells with high proliferative potential were not effectively transduced. Consequently, the majority of registered labeled cells were non-stem stromal cells that survived implantation. Mapping showed that SIs were distributed throughout the mouse genome, with slight preferences to genes. Index-hopping or formation of chimeric molecules was a serious obstacle to the analysis requiring a counting of individual molecules from amplified libraries. Chimeric molecules recombinantly provide false unique molecular identifiers (UMIs), which should be recognized during bioinformatic analysis for proper clustering. Summary/Conclusion: tnPCR measurement provides false positive results from single molecules and cannot be considered as a proper tool for determining marked cell colonies. Non-stem cells of a transplanted stroma contributed to the HEF’s ICM stroma. In the ACL of LTBMC, the efficiency of MSCs infection is noticeably lower than that of more differentiated stromal cells. This may be due to a combination of molecular processes that determine the stem status of a cell. This implies that the efficacy of gene therapy based on such lentiviral vectors may be limited and is determined mainly by the lifetime of infected non-stem cells. On the other hand, this means a reduced risk of stem cell dysfunction. Proper single-cell methods or methods targeting definite subpopulations are required to study stem cells presenting only small subpopulation of cells (10-4-10-6) to evade wrong aggregations. While short-term effects from small subpopulation of stem cells could be insignificant, long-term effects may be shaped by stem cells. Keywords: Insertion site analysis, Mesenchymal stem cell, Mouse model, Lentiviral transduction